The oncolytic effects of reovirus in various cancers have been proven in many clinical trials in human medicine. Oncolytic virotherapy using reovirus for canine cancers is being developed in our laboratory. The objective of this study was to examine the synergistic anti-cancer effects of a combination of reovirus and low doses of various chemotherapeutic agents on mammary gland tumors (MGTs) in dogs. The first part of this study demonstrated the efficacy of reovirus in canine MGTs in vitro and in vivo. Reovirus alone exerted significant cell death by means of caspase-dependent apoptosis in canine MGT cell lines. A single injection of reovirus impeded growth of canine MGT tumors in xenografted mice, but was insufficient to induce complete tumor regression. The second part of this study highlighted the anti-tumor effects of reovirus in combination with low doses of paclitaxel, carboplatin, gemcitabine, or toceranib. Enhanced synergistic activity was observed in the MGT cell line treated concomitantly with reovirus and in all the chemotherapeutic agents except toceranib. In addition, combining reovirus with paclitaxel or gemcitabine at half dosage of half maximal inhibitory concentration (IC50) enhanced cytotoxicity by activating caspase 3. Our data suggest that the combination of reovirus and low dose chemotherapeutic agents provides an attractive option in canine cancer therapy.

Reovirus-suppressed tumor growth in xenograft mouse models of canine MGT. CHMp-5b (1.0 × 107 cells) was implanted subcutaneously at the right flank of mice (day 1). After 14 d, the tumors were treated with a single intratumoral injection of 1.0 × 108 PFUs of reovirus or UV-inactivated reovirus (control). Tumor size was measured with a caliper (A). Data represents the mean ± SD of each treatment group. * indicates P < 0.05. UV-inactivated reovirus did not show any infectivity to L929 cells. B — Hematoxylin and eosin (H&E) and immunohistochemical (IHC) results from representative mice with no treatment, UV-inactivated reovirus, or reovirus 21 d post-transplantation are shown. Antibody used for IHC is targeted at the reoviral μ and σ proteins (,).

The combination of reovirus and chemotherapeutic agents enhanced cell death by means of caspase-dependent apoptosis. Whole cell lysates were prepared from CHMp-5b treated with the chemothera-peutic agents at half of IC50 for 48 h. Proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before the cleavage of caspase 3 was assessed using Western blotting with anti-caspase 3 antibody. Actin was used as protein-loading controls. Results shown represent 3 repeats. Bar graphs shown are densitometric quantifications of the relative expression levels of cleaved caspase 3 of chemotherapeutic agents with or without reovirus relative to cleaved caspase 3 of reovirus alone. Mean ± SD of 3 independent experiments is shown. Statistical analysis was carried out using t-test between reovirus alone and chemotherapeutic agents with or without reovirus. * indicates P < 0.05.